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Biochemical characterization of beta-glucosidase BglX from Escherichia coli

Ngo, Lorna Q.
http://rave.ohiolink.edu/etdc/view?acc_num=ysu1338815851

Abstract Details

2012, Master of Science in Chemistry, Youngstown State University, Department of Chemistry.
Beta-glucosidase BglX from E. coli is an enzyme that hydrolyzes the β(1→4) glycosidic bonds. Unlike other beta-glucosidases produced by E. coli, this enzyme is found in the periplasm of the cell rather than the cytoplasm. BglX is a suitable target for drug therapy since this enzyme is produced solely by bacteria. Identification of key residues in the active site is important in the elucidation of the mechanism of BglX and for rationalization of inhibitor design. The objective of this research is to develop the purification procedure for BglX, characterize BglX, and identify the important residues in the active site of BglX. Purification of BglX was performed with ammonium sulfate precipitation, ion exchange and hydrophobic interaction chromatographies. The kinetic parameters of BglX were analyzed; the enzyme has a KM of 0.35 mM, a kcat of 1.19 s-1, and a catalytic efficiency of 3.4 X 103 M-1s-1 for the chromogenic substrate, para-nitrophenol-β-D-glucopyranoside. Aspartate 287 was selected for mutation based on homology with other glycosidases. BglX mutants were constructed through PCR-based site-directed mutagenesis. The activity of the BglX D287N mutant protein was compared to the wild type BglX protein. Replacement of aspartate 287 with asparagine resulted in significantly reduced glucosidase activity, indicating the importance of this residue in catalysis.
Nina Stourman, PhD (Advisor)
Michael Serra, PhD (Committee Member)
Jonathan Caguiat, PhD (Committee Member)
72 p.
Biochemistry
biochemistry; E. coli

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Citations

  • Ngo, L. Q. (2012). Biochemical characterization of beta-glucosidase BglX from Escherichia coli [Master's thesis, Youngstown State University]. OhioLINK Electronic Theses and Dissertations Center. http://rave.ohiolink.edu/etdc/view?acc_num=ysu1338815851

    APA Style (7th edition)

  • Ngo, Lorna. Biochemical characterization of beta-glucosidase BglX from Escherichia coli. 2012. Youngstown State University, Master's thesis. OhioLINK Electronic Theses and Dissertations Center, http://rave.ohiolink.edu/etdc/view?acc_num=ysu1338815851.

    MLA Style (8th edition)

  • Ngo, Lorna. "Biochemical characterization of beta-glucosidase BglX from Escherichia coli." Master's thesis, Youngstown State University, 2012. http://rave.ohiolink.edu/etdc/view?acc_num=ysu1338815851

    Chicago Manual of Style (17th edition)

ysu1338815851
664
© 2012, all rights reserved.
This open access ETD is published by Youngstown State University and OhioLINK.